The present invention relates to systems for detecting hybridization between targets in a fluid sample and nucleic acid probes disposed on the surface of a nucleic acid probe array.
Nucleic acid probe arrays are used for detecting the presence of various target molecules in a fluid sample. The nucleic acid probes are preferably fabricated directly onto the surface of the probe array using light directed synthesis. As the fluid sample is passed over the surface of the probe array, the target molecules will hybridize with corresponding nucleic acid sequences which are attached to the surface of the probe array.
The target molecules are preferably prepared with tags, such as fluorescin, in order to discriminate areas of strong hybridization to the probes on the array. Specifically, a laser is directed at various discreet locations on the probe array, and fluorescent light is emitted at hybridization locations. By knowing the location of various nucleic acid probe sequences attached to the array (i.e.: when the array was initially fabricated), and by determining the locations at which hybridization has occurred (by detecting fluorescence emissions therefrom), it is then possible to determine whether various target molecules are present in the fluid sample.
Typically, a microscopic scanning system is used to detect the locations of hybridization between the target molecules in fluid sample and the probes on the surface of the probe array. Unfortunately, a disadvantage of optical scanning of the probe array to determine hybridization locations is that it requires a complex scanning and fluorescence detection optical system. As such, precise optics are required which are adapted to discriminate between various microscopically small locations on the surface of the probe array. Moreover, a further disadvantage of detecting hybridization by an optical system is that observation of the behavior of each probe over a range of conditions is often difficult.
The present invention provides systems for detecting locations of hybridization on the surface of a nucleic acid probe array between targets present in a fluid sample and nucleic acid probes disposed on the surface of the probe array.
In a preferred aspect, the present invention comprises measuring the temperature at a plurality of discreet locations on the surface of the probe array while applying an oscillating level of energy to the probe array, thereby causing the temperature of the probe array to oscillate.
By detecting a decreased range of temperature oscillation at least one of a plurality of locations on the probe array, hybridization is detected at the at least one of a plurality of locations on the probe array. Specifically, a decreased range of temperature oscillation at the particular location on the probe array (in response to a steady oscillation of energy applied to the array) is indicative of an increase in heat capacity at that location due to the latent heat of hybridization between at least one target in the fluid sample, and at least one nucleic acid probe disposed at the particular location in question on the surface of the probe array.
At the point of hybridization between the probe and the target molecules, the apparent heat capacity at the probe/target interface will increase. This increased heat capacity can be sensed as a decreased oscillating thermal response. Accordingly, by detecting a spike in the apparent heat capacity at the nucleic acid probe/substrate interface (the surface of the probe array), hybridization can be detected.
In various preferred aspects of the invention, the oscillating level energy applied to the surface of the probe array is applied by a heater disposed under the probe array. Preferably, an array of heaters is disposed under the probe array with each heater being disposed under a small xe2x80x9cpatchxe2x80x9d of probes.
An advantage of the present invention is that the need for optical scanning of the probe array to detect hybridization location is overcome. Consequently, the need to preparer the target molecules with a tag such as fluorescin is also overcome.
Hybridization bond energies between probes which are attached to the probe array and target molecules in the fluid sample may vary widely, thus resulting in widely variant equilibrium constants which may experience some hysteresis as the stringency (temperature or ionic trough) is varied.
Accordingly, an optional advantage of using an array of heaters with each heater being disposed under a patch of probes is that it is possible to adjust the temperature under each patch of probes to a temperature which is approximately equal to the temperature at which hybridization occurs between probes at a particular patch of probes and targets in the fluid sample. An advantage of adjusting the temperature at each patch of probes (by each heater) is that the probe site can be optimized for detecting hybridization. This translates to a higher quality signal, at a larger probe range. As such, a further advantage of the present system is that it may provide a better signal for determining true vs. near matches.
In preferred aspects of the invention, a temperature monitoring system is used to measure the temperature at the plurality of locations on the nucleic acid probe array. This temperature monitoring system may preferably comprise a differential scanning calorimetry system. In alternate aspects of the present invention, an infrared scanner may be used to measure the temperature at a plurality of locations on the surface of the probe array.
In an exemplary aspect of the invention, the heaters are formed on suspended diaphragms of silicon nitrate and the heaters are made of polycrystalline silicon.
In an alternate embodiment, the nucleic acid probe array is disposed over an optically absorbing material (for example, a thin nickel film) which is in turn disposed over a thermal insulation layer. In an exemplary aspect of the invention, this thermal insulation layer comprises a material selected from the group consisting of a ceramic, silicon or glass.
In this alternate embodiment of the present invention, the application of an oscillating level of energy to the surface of the probe array is performed by directing the outputs of first and second lasers at the surface of the probe array. In this embodiment of invention, the first laser is preferably adapted to control the xe2x80x9cgrossxe2x80x9d or a large scale temperature at the probe array with a second laser being adapted to xe2x80x9cfine tunexe2x80x9d the oscillating temperature at the probe array. As such, the second laser acts as a xe2x80x9cprobexe2x80x9d laser, and an infrared scanner is preferably used to detect the transient heating signal from the probe (ie: second) laser.
In yet another embodiment of the present invention, an electrode is positioned in the target liquid, an insulating layer is positioned under the nucleic acid probe array and a silicon n-p-n junction is disposed underneath the insulating layer.
In this alternate embodiment of the invention, a laser beam is directed at the under side of the n-p-n junction, thereby forming a circuit between the n-p-n junction and the electrode in the target liquid. By measuring the impedance of this circuit, hybridization can be detected. In preferred aspects of this invention, the laser beam is scanned back and forth across the underside of the n-p-n junction, thereby measuring the impedance of the resulting circuit at a plurality of discrete locations on the nucleic acid probe array.
An advantage of this embodiment of the present invention is that the laser beam unblocks small localized regions of the n-p-n junction, thus avoiding the problem of parasitic capacitance. As such, hybridization can be detected at small discreet locations on the probe array without interference from hybridization at adjacent locations on the on the probe array. By avoiding the problem of parasitic capacitance, a high spatial resolution can be achieved. Specifically, parasitic capacitance is decreased in the present system by the creation of a lightly doped blocking diode region under the nucleic acid probe array and an optical beam (such as a laser beam) is then be used to unblock this region.
The silicon layer is protected from the target liquid by the insulating layer. In an exemplary aspect, the insulating layer comprises silicon nitride, silicon carbide, diamond-like carbon, or boron nitride.
A depletion region will form in the silicon layer adjacent to the insulating layer. The n-p-n junction forms an additional depletion region which reduces the parasitic capacitance.
Moreover, the AC impedance of the circuit will be sensitive to changes close to the insulating layer within a distance defined as the Debye layer. By scanning the laser light beam across the underside of the system, a local hybridization pattern can be determined.
In accordance with this embodiment of the present invention, hybridization locations may be detected electronically by: 1) detecting a shift in double-layer capacitance resulting from a change in the dielectric constant, 2) detecting a shift in the point of zero charge in a semiconductor electrolyte interface, and 3) detecting a shift in the zeta potential of the insulating layer. The second and third mechanisms preferably require a semiconductor substrate.
In various alternate embodiments, the p-n junction is eliminated; photocurrent is used to measure surface changes, an ion sensitive material is placed over the insulating layer; and/or the device may be defined on an insulator using SOI technology to reduce parasitic capacitance.